The invention is generally in the field of methods and compositions for treating neurodegenerative diseases such as Parkinson""s disease, using viral and non-viral delivery systems that deliver therapeutic agents to specific regions of the brain. More specifically, using an adeno-associated viral vector to deliver a nucleotide sequence encoding glutamic acid decarboxylase (GAD) to specific regions of the brain that are overstimulated or disinhibited in neurodegenerative diseases.
The major inhibitory neurotransmitter in the brain is gamma-aminobutyric acid (GABA), (Roberts et al, GABA in Nervous System Function, Raven Press: New York, 1976; McGeer EG, et al, Glutamine, Glutamate, and GABA in the Central Nervous System; Hertz L, Kvamme E, McGeer E G, Schousbal A, eds., Liss: New York, 1983;3-17). Loss of GABA signaling, by a reduction in release, loss of neurons which synthesize GABA, or antagonism of GABA receptors leads to disinhibition, overexcitation and depending on the specific brain region involved, may result in epilepsy, movement disorders or other neurological deficits and symptoms.
Diseases such as Parkinson""s disease, Huntington""s disease, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig""s Disease), Epilepsy and Alzheimer""s disease, have proved difficult to treat. Few, if any therapies, have proved effective in slowing or arresting the degenerative process associated with these diseases. In Parkinson""s Disease (PD), the primary neurochemical disturbance is believed to be the loss of substantia nigra (SN) dopaminergic (DA) neurons. This loss of DA neurons leads to a profound deficit of DA in the projection areas of the caudate and putamen and results in a loss of signaling through dopamine receptors in the postsynaptic neurons. These neurons, via efferents referred to as the direct and indirect pathways, synapse on other cells in the basal ganglia circuitry. Of most relevance to PD, the loss of dopamine receptors in the basal ganglia circuitry leads to loss of drive in the GABAergic inhibitory input to the subthalamic nucleus.
The loss of inhibitory GABAergic drive to the subthalmic nucleus (STN) results in increased activity of the STN which sends excitatory (glutamatergic) afferents to the ventrial media (VM) thalamus, the substantia nigra pars reticulata (SNPR) and a lesser projection to the pars compacta, as well as other cells within the basal ganglia including the globus pallidus. When the concentration of GABA diminishes below a threshold level in the brain, movement disorders and convulsions may result (See e.g., Karlsson et al, (1974) Biochem. Pharmacol 23:3053-3061). GABA synthesis is regulated by glutamic acid decarboxylase (GAD). GAD is present in the brain as two isoforms, GAD-65 and GAD-67. When the GABA levels rise in the brain the convulsions terminate (See e.g., Hayashi (1959) Physiol. 145:570-578). In convulsive disorders, the reduction in brain GABA levels is often paralleled by a diminished level of GAD (McGeer, et al. GABA in Nervous System Function; Roberts E, Chase T N, Tower D B, eds., Raven Press: New York 1976:487-495; Butterworth et al. (1983) Neurochem. 41:440-447; Spokes et al. (1978) Adv. Exp. Med. Biol. 123:461-473).
Levodopa (L-dopa) has historically been the medication of choice to treat Parkinson""s disease. L-dopa is a precursor to dopamine and is able to cross the blood-brain barrier to target the brain. In order to reduce the global effects of L-dopa, it is often given with carbidopa, a peripheral decarboxylase inhibitor which decreases the metabolism of L-dopa in the peripheral tissues. Unfortunately, the response with L-dopa is not sustainable. Most patients develop adverse effects after long-term usage of L-dopa, and often the benefits of treatment wane as the disease progresses. In addition, several common types of central nervous system dysfunction and peripheral side effects are associated with administration of L-dopa. Toxic side effects to the central nervous system include mental changes, such as confusion, agitation, hallucinations, delusions, depression, mania and excessive sleeping. In addition, L-dopa can exacerbate malignant melanomas or other skin lesions and can have untoward effects in patients with cardiovascular or pulmonary disease, asthma, or renal, hepatic or endocrine disease.
Other methods for treating Parkinson""s disease include transplantation of cells used to repair regions of the brain damaged by neurodegeneration. These cells can be engineered to secrete neuroactive substances such as L-dopa. The procedure typically involves cell transplantation into the stratium. Repair of the damaged regions and secretion of L-dopa depends on the transplanted cells being able to re-establish synaptic connections with several structures situated at a considerable distance from the area of neurodegeneration. However, cell transplantation is a complicated procedure which requires donor tissue, and there have been reports of mortality associated with this procedure.
Alternative forms of treating Parkinson""s disease involve implanting devices for deep-brain stimulation (DBS) in specific regions of the brain. For example, DBS of the STN. These devices are typically electrodes implanted into the STN. The electrode is then stimulated at a desired frequency to reduce the effect of Parkinson""s disease. The significance of the STN overactivity is reflected in the success of ablative surgery of the STN in both animal models of Parkinson""s disease, as well as in human Parkinson""s disease itself. In addition to ablation, implantation of nedtronic stimulators are commonly employed. The mechanism of the stimulators is believed to be mediated by local inhibition (via GABA signaling), and is replicated by the local infusion of GABA agonists.
Each of these approaches, surgical ablation, electrical stimulation and infusion of pharmacological GABA agonists is effective in disease palliation, but each has significant adverse effects. For example, extensive invasive surgery, a high risk of infection and potential damage to the brain and in the case of drug infusion, very transient efficiency.
Thus, the treatments for neurodegenerative disorders are palliative at best, with limited and transient efficacy. Therefore, a need exists for a therapeutic approach which has advantages in targeting specificity, both short and long-term efficacy, as well as neuroprotection, without extensive surgery or side-effects.
The invention is based, at least in part, on the discovery that localized delivery of a vector comprising a therapeutic agent to a specific region of the brain that is overstimulated or disinhibited in neurodegenerative diseases, can reduce the effect of overstimulation and promote the improvement of the neurodegenerative disease. In particular, the invention pertains to methods and compositions used to deliver a vector, (e.g., an adeno-associated virus vector (AAV)) comprising a nucleotide sequence encoding glutamic acid decarboxylase (GAD) to target cells, e.g., the subthalmic nucleus of the basal ganglia.
Particularly preferred methods of delivering the vector to specific regions of the brain are those techniques that are simple, safe, and have a lower risk associated with them than lesioning, electrode implantation or cell transplantation. For example, delivery of the vector using stereotactic microinjection techniques, or delivery of the vector using specialized probes, or percutaneous delivery via disruption of the blood-brain barrier. Delivery of the vector using the method of the invention results in minimal immunological or inflammatory responses within the regions of the brain, thus eliminating the need for immunosupression. After delivery of the vector to a specific region of the brain, regional dispersion and/or diffusion of vector occurs ensuring local distribution of gene and stable gene expression.
The methods and compositions are particularly useful for treating neurodegenerative diseases, such as Parkinson""s disease, Huntington""s disease, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig""s Disease), Alzheimer""s Disease as well as epilepsy.
Accordingly, in one aspect, the invention features a method for treating or reducing a neurodegenerative disease in a subject comprising: identifying a target site in the central nervous system that requires modification;
delivering a vector comprising a nucleotide sequence encoding a glutamic acid decarboxylase (GAD) to the target site in the central nervous system; and
expressing the GAD in the target site in an amount effective to treat or reduce the neurodegenerative disease.
In one embodiment, the vector is a viral vector, and is selected form the group consisting of adenovirus vectors, herpes virus vectors, parvovirus vectors, and lentivirus vectors. In a preferred embodiment, the viral vector is an adeno-associated viral vector.
In another embodiment, the vector is a non-viral vector. In a preferred embodiment, the non-viral vector is a liposome-mediated delivery vector.
In one embodiment, the vector is delivered to a specific target site of the central nervous system. In a preferred embodiment, the vector is delivered using stereotaxic delivery, or delivery using specialized probes. In a preferred embodiment, the target site of the central nervous system is a region of the brain. In another preferred embodiment, the region of the brain is selected from the group consisting of basal ganglia, subthalmic nucleus (STN), pedunculopontine nucleus (PPN), substantia nigra (SN), thalamus, hippocampus, cortex and combinations thereof. In a more preferred embodiment, the region of brain is the subthalmic nucleus (STN).
In one embodiment, the neurodegenerative disease is selected from the group consisting of Parkinson""s disease and related movement disorders, Alzheimer""s disease, senile dementia, Amyloid Lateral Schlerosis (ALS), and epilepsy.
In another aspect, the invention features a method for treating or reducing a Parkinson""s disease in a subject comprising:
identifying one or more regions of the brain that require modification;
delivering a vector comprising a nucleotide sequence encoding a glutamic acid decarboxylase (GAD) to the region of the brain; and
expressing the GAD in the region of the brain an amount effective to treat or reduce Parkinson""s disease.
In yet another aspect, the invention features a vector for expression of GAD in cells of the central nervous system comprising a tissue specific promoter operably linked to a nucleic acid encoding GAD, and a post-transcriptional regulatory element.
In one embodiment, the promoter is specific for central nervous system cells and tissues, such as the cells and tissues of the brain. In a preferred embodiment, the promoter is the neuron specific enolase (NSE) promoter.
The vector also preferably comprises post-transcriptional regulatory elements to enhance expression of the encoded protein. In a preferred embodiment, the post-transcriptional regulatory element is the woodchuck post-transcriptional regulatory element. In another preferred embodiment, the GAD is selected from the group consisting of GAD-65 and GAD-67.